Endometrial cancer is the most common gynecological cancer. G-protein coupled receptor 64 (GPR64) belongs to a family of adhesion GPCRs and plays an important role in male fertility. However, the function of GPR64 has not been studied in endometrial cancer. Our objective is to investigate the role of GPR64 in endometrial cancer.
Trang 1R E S E A R C H A R T I C L E Open Access
G-protein coupled receptor 64 (GPR64) acts
as a tumor suppressor in endometrial
cancer
Jong Il Ahn1,2†, Jung-Yoon Yoo3†, Tae Hoon Kim4, Young Im Kim1,2, Russell R Broaddus5, Ji Yeon Ahn1,2,
Jeong Mook Lim1,2*and Jae-Wook Jeong4*
Abstract
Background: Endometrial cancer is the most common gynecological cancer G-protein coupled receptor 64 (GPR64) belongs to a family of adhesion GPCRs and plays an important role in male fertility However, the function of GPR64 has not been studied in endometrial cancer Our objective is to investigate the role of GPR64 in endometrial cancer Methods: We examined the levels of GPR64 in human endometrioid endometrial carcinoma by immunohistochemistry analysis To determine a tumor suppressor role of GPR64 in endometrial cancer, we used a siRNA loss of function
approach in human endometrial adenocarcinoma cell lines
Results: GPR64 levels were remarkably lower in 10 of 21 (47.62%) of endometrial carcinoma samples compared to control Depletion ofGPR64 by siRNA transfection revealed an increase of colony formation ability, cell proliferation, cell migration, and invasion activity in Ishikawa and HEC1A cells The expression ofConnexin 43 (Cx43), a member of the large family of gap junction proteins, was reduced through activation of AMP-activated protein kinase (AMPK) in
Ishikawa cells withGPR64-deficicy
Conclusions: These results suggest that GPR64 plays an important tumor suppressor role in endometrial cancer
Keywords: Endometrial cancer, Tumor suppressor, GPR64, Connexin 43
Background
Endometrial cancer is the most common gynecologic
malignancy, with an estimated 63,230 new cases in 2018
endometrioid adenocarcinoma, which originates from
endometrial hyperplasia, a proliferative process in the
epithelium, is the abnormal thickening of the lining of
the uterus due to an increase in the number of
endomet-rial glands It is a critical risk factor for endometrioid
endometrial carcinoma [3] Despite most cases being
di-agnosed in the early stages of endometrial cancer, a
subset of these patients have poor outcomes and a high
studies have focused on targeted molecular therapies for controlling endometrial malignancies [5], however they are still insufficient Therefore, it is important to identify molecular mechanisms involved in the development and progression of endometrial cancer
G-protein coupled receptor 64 (GPR64) is a member
of GPCR superfamily, which is crucial for male fertility [6–8] GPR64 was expressed in the proximal epididymis and efferent ductule regions with are responsible for spermatozoa maturation and rete testis fluid reabsorp-tion [6–8] In addition, expression of GPR64 was found
in fibroblast-like synovial cells in osteoarthritis [9] The
mesenchymal neoplasms, and GPR64 induces placental growth factor (PGF) and metalloproteinase (MMP1) ex-pression [10] Loss ofGPR64 in ewing sarcoma cell line leads to decreased PGF and MMP1 expression and reduced
© The Author(s) 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver
* Correspondence: limjm@snu.ac.kr ; jeongj@msu.edu
†Jong Il Ahn and Jung-Yoon Yoo contributed equally to this work.
1 Department of Agricultural Biotechnology, Seoul National University, Seoul
08826, Republic of Korea
4 Department of Obstetrics and Gynecology & Reproductive Biology, College
of Human Medicine, Michigan State University, 400 Monroe Avenue NW,
Grand Rapids, MI 49503, USA
Full list of author information is available at the end of the article
Trang 2cellular growth with induced TRAIL dependent apoptosis
[10] Also,GPR64 knock-down in an ewing sarcoma tumor
model in immune deficient mice, reduced metastasis and
invasiveness to the liver and lung [10] GPR64 can activate
G-proteins GS/Gq when over-expressed in xenopus
endometrioid adenocarcinoma [12] However, the role of
GPR64 in endometrial cancer is unknown
Connexin 43 (Cx43) is a member of the large family of
gap junction proteins [13] Gap junctions are
intercellu-lar plasma membrane proteins that provide for the
ex-change of ions and small molecules between adjacent
channel was localized at the plasma membrane, but not
channel may regulate cell growth by transportation of
calcium ions or other ions between intracellular
cyto-plasm and the extracellular environment [15,16] Other
studies suggest that Cx43 can regulate cell growth
and death by direct interaction with regulated cell
cycle proteins including cyclin A, cyclin D1, p21, and
p27 [17, 18] Dysregulation of gap junction intercellular
communication was to linked several human diseases such
as cancer, cardiac ischemia, Charcot-Marie-Tooth (CMT),
and Visceroatrial Heterotaxia Syndrom (VAH) [19, 20]
Cx43 is ubiquitously expressed in human tissues and
con-trols cell growth and differentiation via multiple
mecha-nisms Attenuation of Cx43 is frequently observed in
cancers, resulting in loss of gap junctional intercellular
cells derived from various tissue types has been shown to
result in restoration of normal cell growth and
of breast cancer cells [24] Moreover, knock-out of Cx43
in mice results in increased susceptibility to chemically
in-duced lung adenomas [25] There is an inverse correlation
between Cx43 expression and tumor grade in endometrial
cancer [26] These observations suggest that Cx43 has a
tumor suppression function and is a potential target in
cancer therapy
In this study, we examined the levels of GPR64 in
human endometrioid endometrial carcinoma To
lines Our results showed a new tumor suppressor role
Methods
Human endometrium samples
The human endometrioid endometrial carcinoma
sam-ples were obtained from The University of Texas MD
Anderson Cancer Center The control endometrial
samples were obtained from hysterectomies (e.g., due to leiomyoma or a uterus prolapse) All patients with endo-metrial carcinoma underwent surgery Twenty four con-trols and 21 endometrial cancer samples (not paired) were fixed in 10% buffered formalin prior to embedding
in paraffin wax
Immunohistochemistry analysis Immunohistochemistry analysis was performed as previ-ously described [27] Uterine cross sections from
mounted on saline-coated slides, deparaffinized and rehydrated in a graded alcohol series For antigen re-trieval, heat-induced epitope retrieval was performed using a pressure cooker with antigen unmasking solution (H− 3300; Vector Laboratories, Burlingame, CA) and then sections were pre-incubated with 10% normal rabbit serum in phosphate-buffered saline (PBS; pH 7.5) then incubated with anti-GPR64 (Sc-69,492; Santa Cruz Bio-technology, Dallas, TX) antibody in PBS supplemented with 10% normal rabbit serum overnight at 4 °C The next day, sections were washed with PBS and incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA) for 1
h at room temperature Immunoreactivity was detected using diaminobenzidine (SK-4100; Vector Laboratories) then counterstained with hematoxylin and coverslipped with permount Imunnostaining was analyzed using microscopy software from NIS Elements, Inc (Nikon Instruments Inc., Melville, NY) A semi-quantitative grading system (H-score) was used to compare the im-munohistochemical staining intensities
Cell culture and siRNA transfection
HEC1A cells were maintained in phenol red–free DMEM/F12 medium (Gibco, Grand Island, NY) contain-ing 0.1 mM sodium pyruvate (Gibco), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin streptomycin (P/S; Gibco) Cells were cultured in monolayer at 37 °C in 5%
(Invitrogen Crop., Carlsbad, CA) prior to in vitro culture
Colony forming assay After siRNA treatment, Ishikawa and HEC1A cells were seeded into 6-well plates at a density of 2 × 102cells per
2 ml cell culture medium and media was changed every
72 h for 14 days Upon completion of culture, the plate wells were washed with PBS, fixed with 4% paraformal-dehyde and permeabilized with 100% methanol Colonies were stained with 1% crystal violet and images were
Trang 3captured via microscopy (Nikon Instruments Inc.) using
software from NIS Elements, Inc
Cell proliferation assay
Cell proliferation was measured with a cell count kit-8
(Dojindo molecular technologies, Kumamoto, Japan)
assay according to the manufacturer’s instructions After
siRNA treatment, 1 × 104cells were seeded into 24-well
plate and cell proliferation was documented every 24 h
for 10 days
Wound healing assay
Cell migration was measured by a wound healing assay
cells After siRNA treatment, cells were incubated for 24
h After 24 h, a pipette tip was used to create a scratch
through the cell monolayer and cells were maintained in
measured after 48 h, via inverted microscopy (Nikon
In-struments Inc.) and distance was determined by Image J
software (National Institute of Health, USA) Cell
migra-tion rate was converted to a percentage that measured
the area compared to directly after the scratch
Invasion assay
For the transwell invasion assay, Ishikawa and HEC1A
cells treated with siRNA were plated in the top chamber
of a matrigel-coated membrane (24-well insert; pore size,
8μm; BD Biosciences) at a density of 2.5 × 105
per 200μl serum-free culture medium and culture medium with
10% serum was used as a chemoattractant in the lower
for 48 h and cells that did not invade through the pore
were removed by a cotton swab Cells on the lower
surface of the membrane were fixed with 4%
paraformal-dehyde and permeabilized with 100% methanol Cells
were stained with 1% crystal violet, and images were
captured via fluorescent microscopy (Nikon Instruments
Inc.) using software from NIS Elements, Inc
Annexin V/PI assay
Apoptotosis was measured with a FITC Annexin V
Apoptosis Detection Kit I (BD Pharmigen, San Diego,
CA) assay according to the manufacturer’s instructions
with cold PBS and resuspend cells in 1X binding buffer
100 ul of the solution with 1 × 105cells was transfered to
a 5 ml culture tube and added 5 ul of FITC Annexin V
and 5 ul PI The cells were gently vortexed and
incu-bated at room temperature in the dark After 15 min,
400 ul of 1X binding buffer was added to culture tube
and analyzed by Flow cytometry (FACScalibur; Becton
Dickinson, San Jose, CA) The data was analyzed using
BD cell/Quest Pro software (Becton Dickinson)
RNA isolation and quantitative RT-qPCR Total RNA was isolated using the RNeasy total RNA iso-lation kit (Qiagen, Valencia, CA) according to the manu-facturer’s instructions As a template for quantitative RT-qPCR, cDNAs were synthesized using quantitative PCR random hexamers and MMLV Reverse
quantified by real-time PCR using a CFX96 Real-time Detection System (Bio-Rad Laboratories, Hercules, CA) and iQ™ SYBR Green Supermix (Bio-Rad Laboratories) RPL7 expression was included in each treatment group for normalization The sequences of the primers used for GPR64 were 5′-CTGCAGGATCCCATTGTCTG-3′ and 5′-TGAAAGGGGTTGAATCTCCC-3′, for Cx43 were 5′-ATGAGCAGTCTGCCTTTCGT-3′ and 5′-TCTGCT TCAAGTGCATGTCC-3′, and for RPL7 were 5′-AAGA AGCGAATTGCTTTGAC-3′ and 5′-CAAATCCTCCAT GCAGATGA-3′
Western blot analysis Western blot analyses were performed as described previously [27] Briefly, twenty five micrograms of protein lysates were electrophoresed via SDS-PAGE and trans-ferred onto polyvinylidene difluoride membrane (Milli-pore Corp., Bedford, MA) The membrane was blocked with Casein (0.5% v/v) prior to exposure to anti-GPR64 (sc-69,492 Santa Cruz Biotechnology), anti-phospho-AMPK (Thr 172, #5235; Cell Signaling, Danvers, MA), anti-AMPK (#2532, Cell Signaling), anti-Cx43 (#3512, Cell Signaling), and anti-β-actin (SC-47778; Santa Cruz Biotechnology) antibodies Immunoreactivity was visual-ized by incubation with a horseradish peroxidase-linked secondary antibody followed by exposure to electro-chemiluminescence reagents (ECL) according to the manufacturer’s instructions (GE Healthcare Biosci-ences, Piscataway, NJ)
Immunofluorescence Immunofluorescence was performed as described previ-ously [27] Ishikawa cells were grown on glass coverslips
growth, coverslips were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% of Triton X-100 (Sigma-Aldrich, St Louis, MO) After further washing, Ishikawa cells were exposed to anti-GPR64 (sc-69,492; Santa Cruz Biotechnology) and anti-Cx43 (#3512; Cell Signaling) antibodes overnight at 4 °C and secondary antibodes for 2 h at room temperature Washed coverslips were then mounted onto microscope slides with a DAPI-impregnated mounting media (Vec-tor Labora(Vec-tories) to enable nuclear visualization, and
Trang 4images were captured via a Zeiss LSM700 confocal
micro-scope (Carl Zeiss microImaging GmBH, Jena, Germany)
using ZEN 2009 software (Carl Zeiss microImaging)
Statistical analysis
Cell experiments were measured in triplicate and
aver-aged to achieve a single value for each combination of
treatment, time point, and cell line Statistical analyses
Tukey’s post hoc multiple range test or Student’s t-tests
using the Instat package from GraphPad (San Diego,
CA).p < 0.05 was considered statistically significant
Results
The levels of GPR64 are altered in a subset of
endometrial cancer
To determine the levels of GPR64 in endometrial cancer,
we performed immunohistochemical analysis using
tis-sue from 24 controls and 21 endometrioid endometrial
carcinoma samples In control endometrium, GPR64
proteins were strongly detected in the nucleus, cytosol
and apical membranes of stromal and epithelial cells of
endometrium from the proliferative phase and secretory
phases in women and the levels of GPR64 proteins were
not significantly changed during the menstrual cycle
(Fig 1a) Interestingly, the levels of GPR64 were
signifi-cantly lower in 47.62% (10/21) of endometrial cancer
tissue compared with controls (p < 0.001) However, levels of GRP64 were unchanged in 52.38% (11/21) of
Epi-didymal tissue was included for the
expression of GPR64 was detected in apical membranes
as well as in some nuclei of epididymal duct epithelial cells (Additional file 1: Figure S1) These results suggest that GPR64 may play a tumor suppressor role in certain cases of endometrial cancer
Attenuation ofGPR64 increases colony forming ability and cell proliferation
The continual unregulated proliferation of cells is essen-tial to cancer development [30, 31] To characterize the proliferative role of GPR64 in endometrial cancer, we performed a colony forming assay and a cell prolifera-tion assay in Ishikawa and HEC1A cells transfected with GPR64 siRNA First, we confirmed the decrease of GPR64 mRNA and protein levels by GPR64 siRNA transfection compared to non-targeting pool siRNA by RT-qPCR and western blot analysis, respectively (Fig.2a and b) The colony formation assay enables us to deter-mine the survival and proliferation of cells [32] Colony formation ability was significantly increased in Ishikawa
A
B
Fig 1 Levels of GPR64 in human endometrial cancer a Representative immunohistochemistry andsemiquantitative analysis for GPR64 expression
in control endometrium from women in proliferative, early, and mid-secretory phases b Representative immunohistochemistry and
semiquantitative analysis for GPR64 expression in endometrial cancer tissues The semiquantitative analysis of immunohistochemistry analysis was calculated by H-score among 24 control endometrium and 21 endometrial cancer tissues High level of GPR64 was observed in 100% control endometrium (6/6) and 52.38% endometrial cancer (11/21), and low level was observed in 47.62% endometrial cancer (10/21) ***, p < 0.001
Trang 5proliferation levels of Ishikawa cells were significantly
GPR64 regulates cell apoptosis using an Annexin V/
PI assay The ratio of apoptotic cells was not different
non-tar-geting pool siRNA transfected cells of Ishikawa and
re-sults suggest that GPR64 suppresses epithelial
prolif-eration of endometrial cancer cells
Attenuation ofGPR64 increases cell migration and
invasion
We examined the effect of GPR64 attenuation in cell
mi-gration and invasion Our wound healing assay revealed
signifi-cantly increased the migration ability of Ishikawa and
HEC1A cells (Fig.3) TheGPR64-deficient Ishikawa cells
recovered the wound over 61% after 48 h, while control
cells healed less than 44% of wound distance (Fig 3a)
wound over 71% but the control cells healed only less than 59% of wound distance (Fig.3b)
infiltration rate in the transwell invasion assay of Ishikawa and HEC1A cells compared to siRNA con-trols Invasion ability of GPR64-deficient Ishikawa cells was increased by more than three-times compared to
knockdown HEC1A cells was increased by more than two-times (Fig.4b)
Attenuation of GPR64 reduces the expression of Connexin 43 trough activation of AMPK
Activation of AMP-activated protein kinase (AMPK) plays a critical role in induction of EMT in multiple
phosphorylation level of AMPK (Thr 172) in Ishikawa
knock-down compared to control, however total AMPK levels
Fig 2 Effect of GPR64 loss on cell growth in human endometrial cancer cells a and b The expression of GPR64 mRNA and protein in Ishikawa (a) and HEC1A (b) cells transfected with non-targeting pool (NT) siRNA or GPR64 siRNA was examined by RT-qPCR and Western blot analysis, respectively c and d Colony formation assay of Ishikawa (c) and HEC1A (d) cells transfected with NT siRNA or GPR64 siRNA Samples from each treatment were transferred to flat-bottomed 24-well plates and incubated Cells were fixed and stained with crystal violet The average colony formation number was quantified with crystal violet stained cells e and f Cell proliferation assay of Ishikawa (e) and HEC1A (f) cells transfected with NT siRNA or GPR64 siRNA The results represent the mean ± SEM *, p < 0.05; **, p < 0.01; and ***, p < 0.001
Trang 6were not different between control and GPR64-deficient
cells (Fig.5a)
Connexin proteins are frequently dysregulated in
tu-mors, resulting in loss of gap junctional intercellular
Cx43 expression exhibited dysfunctional gap junctional
intercellular communication [19,38,39] The expression
and function of Cx43 have been correlated with
carcino-genesis in endometrial cancer [26, 40] Therefore, we
Western Blot analysis Depletion of GPR64 significantly
reduced the levels of Cx43 proteins on day 1 and day 6
compared with non-targeting pool siRNA by Western
mRNA was significantly reduced in the Ishikawa cells
with non-targeting pool siRNA (Fig.5c)
Next, we performed double immunofluorescence for
GPR64 is colocalized with Cx43 in Ishikawa cells
However, the expression of Cx43 protein was
compared to non-targeting pool siRNA
Discussion
G protein-coupled receptors (GPCRs) are transmem-brane receptors and play important roles in multiple biological processes Aberrant expression of these recep-tors has been linked to cancer development and
GPCRs is not known in endometrial cancer The present study shows that the expression of GPR64 was distinctly lower in a subset of endometrioid endometrial carcin-oma These results suggest a tumor suppressor role of GPR64 in endometrial cancer
Recently, Richter et al identified that GPR64 is specif-ically overexpressed in Ewing sarcoma (ES) but is also up-regulated in prostate, kidney, and lung carcinoma [10] This study identified that suppression ofGPR64 in
ES by siRNA, led to impaired colony formation, cell growth, and metastasis in Rag2(−/−)γC(−/−)mice Moreover,
A
B
Fig 3 Ability of cell migration associated with GPR64 expression in endometrial cancer The wound healing assay was performed in Ishikawa (a) and HEC1A (b) cells transfected with NT siRNA or GPR64 siRNA After scratch, cells were incubated for 48 h to determine the cell migration ability Representative result of wound healing assay and quantification of cell migration by wound healing assay during in vitro culture The results represent the mean ± SEM *, p < 0.05
Trang 7suppression of GPR64 induced TRAIL mediated apoptosis
also involved in cell adhesion and migration through
acti-vation of serum response element (SRE) and nuclear factor
kappa-light-chain-enhancer of activated B cells (NFκB) and
its knockdown by siRNA in the highly motile breast cancer
cell lines results in a reduction in cell adhesion and
migra-tion [46] However, our results showed that depletion of
GPR64 increases the cell proliferation, migration, and
inva-sion of endometrial cancer cells These results suggest the
role of GPR64 as a tumor suppressor in endometrial can-cer It remains possible thatGPR64 plays dual functions in different cancer conditions depending on the spatial and
results suggest a new role of GPR64 as a tumor suppressor
in endometrial cancer, but further studies are needed to provide a conclusive answer
Unfortunately, we could not characterize the subtype
of endometrial cancers where GPR64 could be a tumor
A
B
Fig 4 An increase of cell invasion by GPR64 loss in endometrial cancer cells Representative result of transwell invasion assays of Ishikawa (a) and HEC1A (b) cells transfected with NT siRNA or GPR64 siRNA Quantification of invasion through matrigel and transwell membrane in Ishikawa and HEC1A cells with or without GPR64 siRNA treatment The results represent the mean ± SEM **, p < 0.01
Fig 5 The reduction of Cx43 levels by GPR64 loss through AMPK activation in Ishikawa cells a The Western blot analysis of phospho-AMPK Thr172 , Total AMPK, and Cx43 proteins in Ishikawa cells transfected with NT siRNA or GPR64 siRNA on day 1 and day 6 Actin was used as sample-loading control b Quantification of western blot analysis of Cx43 c The expression of Cx43 gene during in vitro culture after GPR64 siRNA transfection Cx43 was significantly decreased on day 3 and day 6 in cells transfected with GPR64 siRNA The results represent the mean ± SEM *, p < 0.05; and
**, p < 0.01
Trang 8suppressor The levels of GPR64 were not correlated
with grade or stage of endometrial endometrioid
adeno-carcinoma which is the most frequently occurring
endo-metrial cancer cell type [47] Cancer classification in the
clinic is primarily based on histological analysis in the
proper clinical context Although highly informative,
histopathology can be hampered by limitation in its
ability to distinguish subtypes of cancers and molecular
signatures Although we could not characterize the
sub-type of endometrial tumors based on GPR64 levels in
this study, composite molecular profiling of tumor
speci-mens is increasingly becoming recognized as an adjunct
to traditional histopathology Therefore, the molecular characterization of tumor types using GPR64 expression may help to identify additional objective tools to en-hance the classification of endometrial cancer However, there is a need to investigate a tumor suppressor role of GPR64 in specific subtypes of endometrial cancer Several adhesion GPCRs have been shown to be involved in cell adhesion and migration, hereby influen-cing tumor progression [46] GPR26 is a potent regulator
of energy homeostasis through controlling hypothalamic
AMPK inhibits Cx43 expression in bladder smooth
different between control and GPR64-deficient cells, the
knock-down compared to control AMPK is associated with cancer development as an important mediator in maintaining cellular energy homeostasis [33, 34], and its activity is increased by extracellular changes such as de-pletion of ATP, low glucose, and changes of NADPH levels [35] Additionally, AMPK inhibits Cx43 expression
in bladder smooth muscle cells [36] Cx43 expression has been associated with a wide variety of cancers, including liver tumor, colon cancer, breast cancer, ovarian carcin-oma, and endometrial cancer [41] Its role in controlling cell motility and polarity contributes to cancer develop-ment and metastasis Especially, Cx43 decreased with poor differentiation of endometrial cancer [26], and Cx43 is considered to be weakened in progression of carcinogen-esis [40, 42] Thus, our results suggest that GPR64 is a tumor suppressor in endometrial cancer by regulating Cx43 expression through regulation of AMPK activity
Conclusions
We found an attenuation of GPR64 expression in a subset of endometrial cancer We demonstrated that
promoting cell proliferation, migration, and invasion in endometrial cancer cells GPR64 regulates the expression
These results suggest that GPR64 acts as a tumor sup-pressor in endometrial cancer
Additional file
Additional file 1: Figure S1 Expression of GPR64 in mouse epididymis The immunohistochemistry for GPR64 was performed in mouse epididymis as a positive control Immunohistochemical staining of mouse epididymis shows membranous and nuclear positivity in epididymal duct epithelial cells Figure S2 Effect of GPR64 on cell apoptosis in human endometrial cancer cells Annexin V/PI assay were performed in Ishikawa (A) and HEC1A (B) cells transfected with with non-targeting pool (NT) siRNA or GPR64 siRNA to determine the effect of GPR64 on cell apoptosis The apoptotic cells were analyzed by Flow cytometry No difference was found between there were no significant difference between NT siRNA and GPR64 siRNA treatments (PPTX 330 kb)
Fig 6 The colocalization of GPR64 with Cx43 in Ishikawa cells The
colocalization of GPR64 (red) and Cx43 (green) were analyzed in
Ishikawa cells transfected with NT siRNA or GPR64 siRNA by
fluorescence microscopy GPR64 overlaps with Cx43, but its
colocalization was affected by reduction of GPR64 Nuclei were
counterstained with DAPI staining
Trang 9CMT: Charcot-Marie-Tooth; Cx43: Connexin 43; DAB: Diaminobenzidine;
ES: Ewing sarcoma; ESR1: Estrogen receptor α; GPCR: G protein-coupled
receptors; GPR64: G-protein coupled receptor 64; MMP: Metalloproteinase;
PBS: Phosphate-buffered saline; PGF: Placental growth factor;
PGR: Progesterone receptor; siRNA: Small interfering RNA; TCF: T-cell factor;
VAH: Visceroatiral Heterotaxia Syndrom
Authors ’ contributions
JIA and JYY conceived and designed the experimental approach, performed
experiments and prepared the manuscript THK, YIK, and JYA performed the
experiments and analyzed the results RRB provided the human endometrial
specimens and performed analysis and interpretation of data JML and JWJ
conceived and designed the experimental approach, performed data analysis
and prepared the manuscript All authors have read and approved the final
version of manuscript.
Funding
Grant numbers and sources of support:
The design, data collection, data analysis, and data interpretation of this
study were supported by Bio-industry Technology Development Program
(IPET312060 –5), Ministry for Food, Agriculture, Forestry and Fisheries, Republic
of Korea (to J.M.L.), and NIH R01 HD084478 (to J.W.J.) The analysis and
inter-pretation of data and writing support of this manuscript were supported by
Basic Science Research Program through the National Research Foundation
of Korea (NRF-2016R1D1A1B03934346), Ministry of Education, Science and
Technology, Republic of Korea (to J.Y.Y.) and Grant Number P50CA098258
from the National Cancer Institute (to R.R.B and T.H.K.).
Availability of data and materials
The raw data available upon reasonable request from the corresponding
authors.
Ethics approval and consent to participate
The study has been approved by Institutional Review Committee of
University of Texas MD Anderson Cancer Center (Houston, TX), and written
informed consent was obtained from all participants.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Department of Agricultural Biotechnology, Seoul National University, Seoul
08826, Republic of Korea.2Research Institute of Agriculture and Life Sciences,
Seoul National University, Seoul 08826, Republic of Korea 3 Department of
Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical
Sciences, Yonsei University College of Medicine, Seoul 03722, Republic of
Korea.4Department of Obstetrics and Gynecology & Reproductive Biology,
College of Human Medicine, Michigan State University, 400 Monroe Avenue
NW, Grand Rapids, MI 49503, USA.5Pathology, University of Texas M.D.
Anderson Cancer Center, Houston, TX 77030, USA.
Received: 9 January 2019 Accepted: 30 July 2019
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